The U.S. catfish industry is valued in excess of $3.0 billion dollars, and catfish production accounts for over 70% of the total U.S. aquaculture production. Aquaculture raised catfish are vulnerable to infections. Losses to stock from these infections reduce productivity and increase consumer costs—greater than $100 million dollars each year.
Immune defense proteins found in the blood and tissue fluids of vertebrates are of two classes: antigen-specific inducible antibodies (or immunoglobulins), and antigen non-specific (or innate) defense molecules that include the antibacterial peptides, which are analogous in many ways to broad spectrum antibiotics (Marchalonis (1977) Immunity in Evolution (Harvard University Press); Hancock (1997) Peptide Antibiotics 349:418–42.) Teleost fish utilize both types of defense molecules to protect against infection. Teleosts produce IgM and IgD classes of antibodies in response to infection (Wilson and Warr (1992) Ann. Rev. Fish Dis. 2:201–2; Warr (1995) Dev. Comp. Immunol. 19:1–12; Wilson et al. (1997) Proc. Natl. Acad. Sci. USA. 94:4593–4597). In addition, teleosts produce and secrete broad spectrum antibacterial agents such as antibacterial peptides, not just enzymes such as lysozyme (Lemaitre et al. (1996) Eur. J. Biochem. 240(1):143–149; Oren and Shai (1996) Eur. J. Biochem. 237:303–3). Nevertheless, these combined defenses are insufficient to completely protect aquacultured fish from disease, and from a practical perspective to the modern commercial fisheries industry, the natural immune response of fish is simply inadequate.
The channel catfish Ictalurus punctatus is an economically significant species, as well as being perhaps the best-studied model for teleost immunity (Clem et al. (1991) in Phylogenesis of Immunological Functions, ed. Warr and Cohen (CRC Press, Boca Raton), pp. 1–13; Miller et al. (1994) J. Immunol. 152:2180–2189; Ghaffari and Lobb (1991) J. Immunol. 146:1037–1046; Graves et al. (1985) J. Immunol. 134:75–85, Warr et al. (1991) Eur. J. Immunogenetics 18:393–379; Warr (1995) Dev. Comp. Immunol. 19:1–12; Wilson et al. (1997) Proc. Natl. Acad. Sci. USA 94:4593–4597). Channel catfish are susceptible to a variety of bacterial infections that can have a devastating effect on the stock of a fish farm. For example, enteric septicemia (caused by Edwardsiella ictaluri) is a recurrent, expensive problem in the catfish industry. Researchers have tried vaccination strategies to protect catfish from enteric septicemia; however, no truly satisfactory E. ictaluri immunization protocol exists. In fact, vaccination strategies have been generally unsuccessful for many catfish diseases (including E. ictaluri), even though they have been successful for other fish species (e.g., cold water vibriosis in Atlantic salmon, Salmo salar; see, Holm and Jorgensen (1987) J. Fish. Dis. 10:85–90.
Catfish farmers have attempted to protect aquacultured fish from disease using three methods. First, they have attempted to vaccinate fish. For example, U.S. Pat. No. 4,287,179 describes immunizing fish against Enteric Redmouth by immersion in water containing killed Y. ruckeri. Second, catfish farmers have attempted to boost the innate immunity of aquacultured fish by administering compounds that activate the immune system non-specifically such as yeast cell wall preparations (Wang and Wang (1997) Comp. Immunol. Microbiol. Infect. Dis 20:261–270). U.S. Pat. No. 5,593,678 describes using protein phosphatase inhibitors prophylactically to protect teleost fish, including catfish, from microbial pathogens. Third, farmers can breed catfish that express transgenes for antibacterial peptides. The transgenic peptides confer non-specific immunity to the fish without exogenous treatments.
Each of these approaches presents difficulties. Embryos and hatchlings cannot be immunized effectively because their immune system has not matured enough to effectively respond to the vaccine. Also, immunization can be a time consuming, labor intensive, and expensive procedure especially when the route of immunization is not via immersion or feeding. Non-specific boosting of the immune system tends to be of short duration, even when it is effective. Transgenic approaches are labor intensive and expensive to develop; however, compared to the other strategies, transgenic fish are likely the best solution in the long term because transgenic fish do not need subsequent treatments to protect against disease.
Antibacterial peptides are well described in the literature. The number of structural families is very large, and it is likely that antibacterial peptides occur ubiquitously among species (Hancock (1997) Peptide Antibiotics 349:418–42; Hoffmann et al. (1996) Curr. Opin. Immunol. 8:8–13; Boman (1996) Scand. J. Immunol. 43:475–482; Bevins and Zasloff (1990) Annu. Rev. Biochem. 591:395–414; and Lehrer et al. (1993) Annu. Rev. Immunol. 11:105–128). Among the earliest discovered and best-studied antibacterial peptides are the cecropins, small cationic peptides originally characterized in the moth Hyalophora cecropia (Steiner et al. (1981) Nature 292:246–248). The cecropins are both bacteriocidal and bacteriostatic. In fact, researchers have demonstrated that cecropins can inhibit or kill other types of pathogens including virus, fungi, and protozoa.
Cecropins possess a number of key characteristics that are likely to be useful in aquaculture. The peptides exhibit a broad spectrum of activity against gram negative bacteria including most of the major bacterial pathogens of catfish (Kelly et al. (1990) J. Fish Dis. 13:317–321; Thune (1993) Fish Medicine, ed. Stoskopf (Saunders Co., Philadelphia), pp. 511–520). The peptides are nontoxic to eukaryotic cells (Jaynes et al. (1989) Pept. Res. 2:157–160). Cecropins are found ubiquitously, and sources include insects and mammals, such as the pig (Lee et al. (1989) Proc. Natl. Acad. Sci. USA 86:9159–9162). Finally, the peptides are well described in the literature, including their physicochemical properties and mode of action (Christensen et al. (1988) Proc. Natl. Acad. Sci. USA 85:5072–5076), which eliminates or reduces the need for experimentation.
Experimental evidence supports the utility of cecropins in aquaculture systems. Passively administered cecropin derivatives can protect against infection with Edwardsiella ictaluri (Kelly et al. (1993) J. App. Aquaculture 3:25–34), a major pathogen of cultured catfish (Thune (1993) in Fish Medicine, ed. Stoskopf (Saunders Co., Philadelphia), pp. 511–520).
Creating a catfish expressing an antibacterial peptide transgene such as cecropin has several potential advantages over conventional immunization strategies. First, the fish will express cecropin from early in development, long before the immune system has matured enough to respond to immunization. Second, a cecropin transgene can confer immunity against a broad range of pathogens, obviating the need for multiple pathogen-specific vaccination preparations and treatments. Cecropin-transgenic catfish thus alleviate two major commercial losses, the destruction of stock due to diseases, and the need for ongoing prophylactic therapy or immunization for healthy stock.
Transgenic fish are the subject of several U.S. patents. For example, U.S. Pat. No. 6,207,817 relates to DNA sequences of fish insulin-like growth factor II (IGF-II) promoter regions and recombinant IGF-II promoters, and the expression of IGF-II promoter regions and recombinant IGF-II promoters in eukaryotic cells and fish embryos.
U.S. Pat. No. 6,015,713 relates to a transgenic fish that expresses human insulin and to the uses of the transgenic insulin in the treatment of diabetes. Notably, although the fish carries a humanized insulin transgene, the fish insulin regulatory sequences that drive expression of the transgene were not modified.
U.S. Pat. No. 5,545,808 describes a transgenic salmonid fish expressing exogenous salmonid growth hormone. This patent claims a method of increasing the growth rate of salmonid fish comprising the steps of a) introducing into the germ line of a salmonid fish a gene encoding a salmonid growth hormone operably linked to a type 3 antifreeze protein promoter and b) culturing the salmonid fish under conditions wherein the salmonid fish expresses the growth hormone gene at levels that increase the rate of growth at least four times over non-transgenic controls.
Sockeye salmon growth hormone genes Types 1 and 2, and sockeye histone and metallothionein gene promoters have been isolated and sequenced as described in U.S. Pat. No. 5,998,697. Terminal sequences for the growth hormone gene were also disclosed. Vectors containing these promoter and terminal sequences (and intermediate sequences) were used to transform fish egg cells, then the transformed fish egg cells were grown into transgenic fish.
U.S. Pat. No. 5,719,055 describes a transposon-based vector, which enhances the integration of DNA into a host genome, particularly a eukaryotic genome. This vector has been used to transform mammalian and fish cells with a non-constitutively expressing transgene coding for cecropin B.
U.S. Pat. No. 6,156,568 discloses transformed animals, transformed animal cells capable of expressing exogenous lytic peptides, genes in eukaryotic cells controlled by exogenous promoters that are responsive to inducers of acute phase proteins, and transposon-based transformation vectors. The patent specifically claims a eukaryotic cell in vitro that contains a gene under the control of the wild-type cecropin B promoter, wherein the promoter is exogenous to the cell. The cells, as recited, are vertebrate and mammalian cells.
U.S. Pat. No. 5,998,698 claims a transgenic catfish having a gene encoding cecropin B operably linked to the native cecropin B promoter, where the cecropin B promoter functions to direct expression of the cecropin B gene; and where the expression of the cecropin B gene imparts resistance to pathogenic bacteria. The patent further claims a transgenic koi and bony fish having a gene encoding cecropin B.
U.S. Pat. No. 6,156,568 discloses a transgenic fish having a moth cecropin gene encoding cecropin B operably linked to the native moth cecropin B promoter. The transgenic fish were protected from a challenge with E. ictaluri. 
Thus attempts have been made to solve the problem of disease infestation by breeding fish that express transgenes coding for anti-pathogenic proteins that kill pathogens. However, this solution has created a second problem. Transgenes are routinely expressed under a viral promoter, but because consumers are likely to perceive that fish carrying viral “fragments” are unhealthy or unsafe, consumers are unlikely to buy them despite the Food and Drug Administration's approval. Therefore, fish farmers are placed in an intractable dilemma of either being forced to bear the costs of high disease incidence or, in the event they choose to farm transgenic disease-resistant fish, being unable to sell the disease-resistant fish due to consumer perceptions.
Methods for producing transgenic fish with enhanced disease resistance and which meet with consumer and FDA approval are needed.